Bioconjugated Oligonucleotides: Recent Developments and Therapeutic Applications.

Oligonucleotide-based agents have the potential to treat or cure almost any disease, and are one of the key therapeutic drug classes of the future. Bioconjugated oligonucleotides, a subset of this class, are emerging from basic research and being successfully translated to the clinic. In this Review, we first briefly describe two approaches for inhibiting specific genes using oligonucleotides-antisense DNA (ASO) and RNA interference (RNAi)-followed by a discussion on delivery to cells. We then summarize and analyze recent developments in bioconjugated oligonucleotides including those possessing GalNAc, cell penetrating peptides, α-tocopherol, aptamers, antibodies, cholesterol, squalene, fatty acids, or nucleolipids. These novel conjugates provide a means to enhance tissue targeting, cell internalization, endosomal escape, target binding specificity, resistance to nucleases, and more. We next describe those bioconjugated oligonucleotides approved for patient use or in clinical trials. Finally, we summarize the state of the field, describe current limitations, and discuss future prospects. Bioconjugation chemistry is at the centerpiece of this therapeutic oligonucleotide revolution, and significant opportunities exist for development of new modification chemistries, for mechanistic studies at the chemical-biology interface, and for translating such agents to the clinic.

FXYD2 antisense oligonucleotide provides an efficient approach for long-lasting relief of chronic peripheral pain.

Chronic pain, whether of inflammatory or neuropathic origin, affects about 18% of the population of developed countries, and most current treatments are only moderately effective and/or cause serious side effects. Therefore, the development of novel therapeutic approaches still represents a major challenge. The Na,K-ATPase modulator FXYD2 is critically required for the maintenance of neuropathic pain in rodents. Here, we set up a therapeutic protocol based on the use of chemically modified antisense oligonucleotides (ASOs) to inhibit FXYD2 expression and treat chronic pain. We identified an ASO targeting a 20-nucleotide stretch in the FXYD2 mRNA that is evolutionarily conserved between rats and humans and is a potent inhibitor of FXYD2 expression. We used this sequence to synthesize lipid-modified forms of ASO (FXYD2-LASO) to facilitate their entry into dorsal root ganglia neurons. We established that intrathecal or intravenous injections of FXYD2-LASO in rat models of neuropathic or inflammatory pain led to a virtually complete alleviation of their pain symptoms, without causing obvious side effects. Remarkably, by using 2'-O-2-methoxyethyl chemical stabilization of the ASO (FXYD2-LASO-Gapmer), we could significantly prolong the therapeutic action of a single treatment up to 10 days. This study establishes FXYD2-LASO-Gapmer administration as a promising and efficient therapeutic strategy for long-lasting relief of chronic pain conditions in human patients.

Electropolymerizable Thiophene-Oligonucleotides for Electrode Functionalization.

Inserting complex biomolecules such as oligonucleotides during the synthesis of polymers remains an important challenge in the development of functionalized materials. In order to engineer such a biofunctionalized interface, a single-step method for the covalent immobilization of oligonucleotides (ONs) based on novel electropolymerizable lipid thiophene-oligonucleotide (L-ThON) conjugates was employed. Here, we report a new thiophene phosphoramidite building block for the synthesis of modified L-ThONs. The biofunctionalized material was obtained by direct electropolymerization of L-ThONs in the presence of 2,2'-bithiophene (BTh) to obtain a copolymer film on indium tin oxide electrodes. In situ electroconductance measurements and microstructural studies showed that the L-ThON was incorporated in the BTh copolymer backbone. Furthermore, the covalently immobilized L-ThON sequence showed selectivity in subsequent hybridization processes with a complementary target, demonstrating that L-ThONs can directly be used for manufacturing materials via an electropolymerization strategy. These results indicate that L-ThONs are promising candidates for the development of stable ON-based bioelectrochemical platforms.

Hydrogel based lipid-oligonucleotides: a new route to self-delivery of therapeutic sequences.

Synthetic OligoNucleotides (ON) provide promising therapeutic tools for controlling specifically genetic expression in a broad range of diseases from cancers to viral infections. Beside their chemical stability and intracellular delivery, the controlled release of therapeutic sequences remains an important challenge for successful clinical applications. In this work, Lipid-OligoNucleotide (LON) conjugates stabilizing hydrogels are reported and characterized by rheology and cryo-scanning electron microscopy (cryo-SEM). These studies revealed that lipid conjugation of antisense oligonucleotides featuring partial self-complementarity resulted in entangled pearl-necklace networks, which were obtained through micelle-micelle interaction driven by duplex formation. Owing to these properties, the Lipid AntiSense Oligonucleotide (LASO) sequences exhibited a prolonged release after subcutaneous administration compared to the non-lipidic antisense (ASO) one. The LASO self-assembly based hydrogels obtained without adjuvant represent an innovative approach for the sustained self-delivery of therapeutic oligonucleotides.

Bolaamphiphile-based supramolecular gels with drugs eliciting membrane effects.

Supramolecular chemistry has garnered important interest in recent years toward improving therapeutic efficacy via drug delivery approaches. Although self-assemblies have been deeply investigated, the design of novel drugs leveraging supramolecular chemistry is less known. In this contribution, we show that a Low Molecular Weight Gel (LMWG) can elicit cancer cell apoptosis. This biological effect results from the unique supramolecular properties of a bolaamphiphile-based gelator, which allow for strong interaction with the lipid membrane. This novel supramolecular-drug paradigm opens up new possibilities for therapeutic applications targeting membrane lipids.

Antisense Oligonucleotide-Based Therapeutic against Menin for Triple-Negative Breast Cancer Treatment.

The tumor suppressor menin has dual functions, acting either as a tumor suppressor or as an oncogene/oncoprotein, depending on the oncological context. Triple-negative breast cancer (TNBC) is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2) and is often a basal-like breast cancer. TNBC is associated with a dismal prognosis and an insufficient response to chemotherapies. Previously, menin was shown to play a proliferative role in ER-positive breast cancer; however, the functions of menin in TNBC remain unknown. Here, we have demonstrated that menin is expressed in various TNBC subtypes with the strongest expression in the TNBC Hs 578T cells. The depletion of menin by an antisense oligonucleotide (ASO) inhibits cell proliferation, enhances apoptosis in Hs 578T cells, highlighting the oncogenic functions of menin in this TNBC model. ASO-based menin silencing also delays the tumor progression of TNBC xenografts. Analysis of the menin interactome suggests that menin could drive TNBC tumorigenesis through the regulation of MLL/KMT2A-driven transcriptional activity, mRNA 3'-end processing and apoptosis. The study provides a rationale behind the use of ASO-based therapy, targeting menin in monotherapy or in combination with chemo or PARP inhibitors for menin-positive TNBC treatments.

Nucleoside-Lipid-Based Nanoparticles for Phenazine Delivery: A New Therapeutic Strategy to Disrupt Hsp27-eIF4E Interaction in Castration Resistant Prostate Cancer.

Heat shock protein 27 (Hsp27) has an established role in tumor progression and chemo-resistance of castration-resistant prostate cancer (CRPC). Hsp27 protects eukaryotic translation initiation factor 4E (eIF4E) from degradation, thereby maintaining survival during treatment. Phenazine derivative compound #14 was demonstrated to specifically disrupt Hsp27/eIF4E interaction and significantly delay castration-resistant tumor progression in prostate cancer xenografts. In the present work, various strategies of encapsulation of phenazine #14 with either DOTAU (N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate) and DOU-PEG2000 (5'-PEG2000-2',3'-dioleoyluridine) nucleolipids (NLs) were developed in order to improve its solubilization, biological activity, and bioavailability. We observed that NLs-encapsulated phenazine #14-driven Hsp27-eIF4E interaction disruption increased cytotoxic effects on castration-resistant prostate cancer cell line and inhibited tumor growth in castration-resistant prostate cancer cell xenografted mice compared to phenazine #14 and NLs alone. Phenazine #14 NL encapsulation might represent an interesting nanostrategy for CRPC therapy.

Analysis of lipid-oligonucleotide conjugates by cyclodextrin-modified capillary zone electrophoresis.

Lipid-oligonucleotide (LONs) based bioconjugates represent an emerging class of therapeutic agents, allowing the delivery of therapeutic oligonucleotide sequences. The LON development requests accurate and efficient analytical methods. In this contribution, LON analysis methods were developed in cyclodextrin-modified capillary zone electrophoresis (CD-CZE). The LONs selected in this study feature different structures, including i) the oligonucleotide length (from 10 to 20 nucleotides), ii) the inter-nucleotide linkage chemistry (phosphodiester PDE or phosphorothioate PTO), and iii) the lipidic part: single- (LONsc) or double-chain (LONdc) lipids. In CD-CZE, the effect of several parameters on the electrophoretic peaks was investigated (buffer, CD, and capillary temperature). The binding interaction between LON and Me-ß-CD was studied in affinity capillary electrophoresis and revealed a 1:1 LON:CD complex. Non-linear regression and three usual linearization methods (y-reciprocal, x-reciprocal, and double-reciprocal) were used to determine the binding constants (K values of 2.5.104 M-1 and 2.0.104 M-1 for LON PDE and LON PTO, respectively). Quantitative methods with good performances and analysis time lower than 5 min were achieved. Importantly, the developed analysis allows a separation between the i) full-length sequence LONs and their truncated sequences, (n-1), (n-2), and (n-4)-mers and ii) LONsc, LONdc and their corresponding unconjugated oligonucleotides. This work highlights the interest of CD-CZE methods for LON analysis.

Lipid oligonucleotides as a new strategy for tackling the antibiotic resistance.

Antibiotic resistance has become a major issue in public health especially for one of the most used antibiotics; the third-generation cephalosporins. One of the main resistance mechanisms in Enterobacteriaceae, is the production of Extended-Spectrum ß-lactamases. Here, we demonstrated that the oligonucleotide therapy is an efficient approach to reduce the resistance of bacteria to antibiotic treatment. Lipid oligonucleotides (LONs) were proved to be efficient strategies in both delivering the oligonucleotide sequences in the prokaryotic cells and decreasing the Minimum Inhibitory Concentration of resistant bacteria to a third generation cephalosporin, the ceftriaxone. Accordingly, we demonstrated the strong antimicrobial potential of this LON strategy targeting the ß-lactamase activity on both clinical and laboratory strains. Our results support the concept that the self-delivery of oligonucleotide sequences via lipid conjugation may be extended to other antimicrobial drugs, which opens novel ways to struggle against the antibiotic resistance.

Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery.

Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 µM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 µM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

Lipid-oligonucleotide conjugates improve cellular uptake and efficiency of TCTP-antisense in castration-resistant prostate cancer.

Translationally controlled tumor protein (TCTP) has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. Therefore, TCTP is now recognized as a potential therapeutic target in several cancers including prostate, breast and lung cancers. We previously showed that TCTP is overexpressed in castration-resistant prostate cancer (CRPC), and it has been implicated resistance to treatment. Recently, we developed TCTP antisense oligonucleotides (ASOs) to inhibit TCTP expression. However, the intracellular delivery and silencing activity of these oligonucleotides remains a challenge, and depend on the use of transfection agents and delivery systems. Here we show that lipid-modified ASO (LASOs) has improved penetration and efficiency in inhibiting TCTP expression in the absence of additional transfection agents, both in vitro and in vivo. Transfection with TCTP-LASO led to rapid and prolonged internalization via macropinocytosis, TCTP downregulation and significant decreased cell viability. We also show that lipid-modification led to delayed tumor progression in CRPC xenografts models, with no significant toxic effects observed.

pH-Cleavable Nucleoside Lipids: A New Paradigm for Controlling the Stability of Lipid-Based Delivery Systems.

Lipid-based delivery systems are an established technology with considerable clinical acceptance and several applications in human. Herein, we report the design, synthesis and evaluation of novel orthoester nucleoside lipids (ONLs) for the modulation of liposome stability. The ONLs contain head groups with 3'-orthoester nucleoside derivatives featuring positive or negative charges. The insertion of the orthoester function in the NL structures allows the formation of pH-sensitive liposomes. ONL-based liposomes can be hydrolyzed to provide nontoxic products, including nucleoside derivatives and hexadecanol. To allow the release to be tunable at different hydrolysis rates, the charge of the polar head structure is modulated, and the head group can be released at a biologically relevant pH. Crucially, when ONLs are mixed with natural phosphocholine lipids (PC), the resultant liposome evolves toward the formation of a hexadecanol/PC lamellar system. Biological evaluation shows that stable nucleic acid lipid particles (SNALPs) formulated with ONLs and siRNAs can effectively enter into tumor cells and release their nucleic acid payload in response to an intracellular acidic environment. This results in a much higher antitumor activity than conventional SNALPs. The ability to use pH-cleavable nucleolipids to control the stability of lipid-based delivery systems represents a promising approach for the intracellular delivery of drug cargos.